The present invention relates to a method of accurately assaying calcineurin-inhibiting immunosuppressants, such as FK506 and cyclosporin A, which method can be used in the field of medicine.
It is well known that a compound represented by the structural formula and chemical name shown below and also designated as FK506 or FR-900506 has potent immunosuppressive activity and can be used as a prophylactic or therapeutic agent for organ transplant rejection or autoimmune diseases (for example, EP-0184162-A2). 
Chemical name: 17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone
As a result of investigations of the mechanism of immunosuppressive activity of FK506, said activity is supposed to be displayed as follows. FK506 binds to FKBP-12, which is a cytoplasmic FK506-binding protein, followed by association with calcineurin, together with calmodulin and calcium ion, to form a complex (FK506:FKBP-12: calcineurin=1:1:1), whereby the phosphatase activity of calcineurin is inhibited. This phosphatase inhibition leads to suppression of the activation of nuclear factor of activated T cells (NFAT) and to inhibition of IL2 production, whereby immunosuppression is caused.
Cyclosporin A, which has similar immunosuppressive activity, is also supposed to show its immunosuppressive activity by forming a similar complex [cf. for example, Biochemical and Biophysical Research Communications, 192 (3), 1388-1394 (1993) and Angew. Chem., Int. Ed. Engl., 31 (1992) 384-400].
The utility of FK506 as an immunosuppressant has been amply studied and, in Japan, FK506 is already on the market as a rejection reaction suppressant particularly in liver transplantation.
However, since its activity is very potent, the determination of an optimum dose is an important issue. It is very important to administer it at a dose with displaying its effective immunosuppressive activity and without producing adverse effects or the like.
For that purpose, several assay methods have been proposed, including an enzyme immunoassay method with an antibody recognizing the antigenic determinant of FKS506 (e.g. EP-0293892-A2), a method in which the above-mentioned enzyme immunoassay is combined with HPLC, and a radioreceptor method utilizing an FK506-binding protein (FKBP-12) [cf. for example, Clin. Chem., 38/7, 1307-1310 (1992)].
On the other hand, studies on the mechanism of its metabolism have revealed that while FK506 undergoes metabolism in the living body, some of its metabolites still retain immunosuppressive activity, and others are capable of binding to a monoclonal antibody to FK506 with only weak immunosuppressive activity [e.g. Drug Metabolism and Disposition, 21 (6), 971-977 (1993)].
Furthermore, the existence of a substance (e.g. 506BD) capable of binding to FK506 binding proteins (FKBPs) but having no immunosuppressive activity has been revealed [e.g. Angew. Chem., Int. Ed. Engl., 31 (1992) 384-400].
Therefore, the previous assay methods using, as an index, either the binding of FK506 to an antibody recognizing the antigenic determinant of FK506, or the binding of an FKBP to FK506, can hardly be said to be capable of accurately measuring the actual state of immunosuppression. The development of an assay method capable of accurately measuring the total concentration of active drug substances including metabolites actually having immunosuppressive activity has been awaited.
Giving their attention to the fact that an immunosuppressant, such as FK506 or cyclosporin A, binds to an immunophilin (protein capable of binding to an immunosuppressant; e.g. FKBP-12 or cyclophilin) and then form a complex with calcineurin, calmodulin and calcium ion and thereby inhibits the activity of calcineurin, the inventors of the present invention succeeded in establishing a method of assaying immunosuppressants making use of the complex forming ability of said substances.
The present invention thus provides a method of assaying immunosuppressants having calcineurin-inhibiting activity, which comprises assaying a complex comprising (1) an immunophilin, (2) calcineurin, (3) calmodulin, (4) calcium ion and (5) an immunosuppressant having calcineurin-inhibiting activity.
In the following, the particular terms used herein within the scope of the present invention are defined and explained in detail and preferred examples are given.
The xe2x80x9cimmunophilinxe2x80x9d means a cytoplasmic receptor protein to which an immunosuppressant binds and includes, for example, FKBP-12 which is an FK506 binding protein having a molecular weight of about 12K and peptidyl prolyl cis-trans isomerase (PPIase) activity, and cyclophilin which is an intracellular receptor for cyclosporin A and has similar PPIase activity and a molecular weight of about 17K. Preferred are FKBP-12 and cyclophilin produced by mammals such as cattle or humans.
They are already known and can be obtained in the same manner as described in J. Am. Chem. Soc., 113, 1409-1411 (1991), Proc. Natl. Acad. Sci. USA, 88, 6229-6233 (1991), Nature, 346, 671-674 (1991), WO 92/01052, WO 91/17439, Nature, 337, 473-475, 476-478 (1989), or Japanese Kokai Tokkyo Koho Hei 02-209897, for instance.
xe2x80x9cCalcineurinxe2x80x9d is known as a calcium ion- and calmodulin-dependent serine-threonine phosphatase, and calcineurin obtained from mammals such as rats, cattle or humans can be used. Rat or bovine calcineurin, for example, is known to be a heterodimer composed of A and B subunits and it is further known that the A subunit includes two isoforms, Axcex1 and Axcex2. Rat calcineurin can be isolated and purified, for example, from the rat brain [cf. for example, J. Neurochem., 58, 1643-1651 (1992)]. Bovine calcineurin can be obtained in the same manner as described in Adv. Enzymol., 61, 149-200 (1989) or Methods Enzymol., 102, 244-256 (1983). It is also commercially available from Upstate Biotechnology Co. Ltd or Sigma Co. Ltd under the product name xe2x80x9cprotein phosphatase 2Bxe2x80x9d, for instance, and such product may also be used.
xe2x80x9cCalmodulinxe2x80x9d is a substance known as a calcium binding protein and is known to activate various enzymes including the above-mentioned calcineurin in the presence of the calcium ion. Calmodulin derived from mammals such as cattle or humans can be used. Bovine calmodulin, for instance, can be prepared and obtained as described in J. Biochem., 87, 1313-1320 (1980), and a commercial product available from Upstate Biotechnology Co. Ltd or Sigma Co. Ltd can also be used.
The xe2x80x9cimmunosuppressant having calcineurin-inhibiting activityxe2x80x9d to be assayed in accordance with the present invention means a compound which inhibits the phosphatase activity of calcineurin by forming a complex with an immunophilin, calcineurin, calmodulin and calcium ion, and has immunosuppressive activity. Preferred examples are compounds of the following formula: 
(wherein R1 is hydroxy or protected hydroxy, R2 is hydrogen, hydroxy or protected hydroxy, R3 is methyl, ethyl, propyl or allyl, R4 is hydroxy or methoxy, R5 is hydrogen, or R4 and R5 together form oxo, n is an integer of 1 or 2, and the symbol comprising a solid line and a dotted line means a single bond or a double bond, provided that when R4 is hydroxy and R5 is hydrogen or when R4 and R5 together form oxo, R2 is not protected hydroxy.)
The term xe2x80x9clowerxe2x80x9d as used in defining the symbols used in the above general formula [I], and in the subsequent description means, unless otherwise indicated, that the number of carbon atoms is 1 to 6.
Suitable protective groups for use in the xe2x80x9cprotected hydroxyxe2x80x9d include the following: 1-(lower alkylthio) (lower) alkyl groups such as lower alkylthiomethyl (e.g. methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, hexylthiomethyl, etc.), and the like, more preferably C1-C4 alkylthiomethyl, most preferably methylthiomethyl; trisubstituted silyl groups such as tri (lower) alkylsilyl (e.g. trimethylsilyl, triethylsilyl, tributylsilyl, tert-butyl-dimethylsilyl, tri-tert-butylsilyl, etc.), lower alkyl-diarylsilyl (e.g. methyl-diphenylsilyl, ethyl-diphenylsilyl, propyl-diphenylsilyl, tert-butyl-diphenylsilyl, etc.), and the like, more preferably tri(C1-C4)alkylsilyl and C1-C4 alkyldiphenylsilyl, most preferably tert-butyl-dimethylsilyl and tert-butyl-diphenylsilyl; acyl groups such as aliphatic acyl, aromatic acyl and aromatic group-substituted aliphatic acyl which are derived from carboxylic, sulfonic and carbamic acids; and the like.
As more specific examples of the above-mentioned acyl groups, there may be mentioned the following:
The aliphatic acyl may include lower alkanoyl which may have one or more suitable substituent(s) such as carboxy (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, carboxypropionyl, carboxybutyryl, carboxyhexanoyl, etc.), cyclo (lower) alkoxy (lower) alkanoyl which may have one or more suitable substituent(s) such as lower alkyl (e.g. cyclopropyloxyacetyl, cyclobutyloxypropionyl, cycloheptyloxybutyryl, menthyloxyacetyl, menthyloxypropionyl, menthyloxybutyryl, menthyloxypentanoyl, menthyloxyhexanoyl, etc.), camphorsulfonyl, lower alkylcarbamoyl having one or more suitable substituent(s) such as carboxy or protected carboxy, for example carboxy(lower)alkylcarbamoyl (e.g. carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl, etc.), protected carboxy(lower)alkylcarbamoyl such as tri(lower)alkylsilyl(lower)alkoxycarbonyl(lower)alkylcarbamoyl (e.g. trimethylsilylmethoxycarbonylethylcarbamoyl, trimethylsilylethoxycarbonylpropylcarbamoyl, triethylsilylethoxycarbonylpropylcarbamoyl, tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl, trimethylsilylpropoxycarbonylbutylcarbamoyl, etc.), and the like.
The aromatic acyl may include aroyl which may have one or more suitable substituent(s) such as nitro (e.g. benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, nitronaphthoyl, etc.), arenesulfonyl which may have one or more suitable substituent(s) such as halogen (e.g. benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, iodobenzenesulfonyl, etc.), and the like.
The aromatic group-substituted aliphatic acyl may include ar(lower)alkanoyl which may have one or more suitable substituent(s) such as lower alkoxy and trihalo(lower)alkyl (e.g. phenylacetyl, phenylpropionyl, phenylbutyryl, 2-trifluoromethyl-2-methoxy-2-phenylacetyl, 2-ethyl-2-trifluoromethyl-2-phenylacetyl, 2-trifluoromethyl-2-propoxy-2-phenylacetyl, etc.), and the like.
More preferred acyl groups among the above-mentioned acyl groups are C1-C4 alkanoyl which may have carboxy, cyclo(c5-C6)alkyloxy(C1-C4)alkanoyl having two (C1-C4)alkyl groups on the cycloalkyl moiety, camphorsulfonyl, carboxy(C1-C4)alkylcarbamoyl, tri(C1-C4)alkylsilyl (C1-C4)alkoxycarbonyl(C1-C4)alkylcarbamoyl, benzoyl which may have one or two nitro groups, benzenesulfonyl having halogen, phenyl(C1-C4)alkanoyl having C1-C4 alkoxy and trihalo(C1-C4)alkyl. Most preferred among these are acetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and 2-trifluoromethyl-2-methoxy-2-phenylacetyl, and the like.
The compounds represented by the above general formula [I] and a method of production thereof are already known, and said compounds may be obtained as similar manner to that of described in EP-0184162-A2 and EP-0353678-A2.
In particular, those compounds that are designated as FR900506 (=FK506), FR900520, FR900523 and FR900525 are produced by a microorganism of the genus Streptomyces, particularly Streptomyces tsukubaensis No. 9993 (FERM BP-927) or Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 (FERM BP-928), as described in EP-0184162-A2.
Furthermore, cyclosporins, such as cyclosporin A, B, C, D, E, F and G, or derivatives thereof, which are already known as described, for example, in U.S. Pat. Nos. 4,117,118, 4,215,199, 4,288,431 and 4,388,307, Helv. Chim. Acta, 60, 1568 (1977) and 65, 1655 (1982), and Transplant. Proc., 17, 1362 (1985), are also capable of complex formation with a cyclophilin, calcineurin, calmodulin and calcium ion and are therefore immunosuppressants having calcineurin-inhibiting activity, hence can be assayed by the assay method of the present invention.
The method of forming the complex comprising an immunophilin, calcineurin, calmodulin, calcium ion and an immunosuppressant having calcineurin-inhibiting activity in the practice of the present invention has no particular characteristic features and the complex can be formed by allowing a sample containing the immunosuppressant having calcineurin-inhibiting activity to react with an immunophilin, calcineurin, calmodulin and calcium ion in an appropriate solution in the conventional manner.
Thus, for example, it will suffice that the respective components be allowed to stand in an appropriate solution under warming at 30 to 40xc2x0 C. for several hours.
In practicing said assay method actually in the field of medicine, the term xe2x80x9csample containing an immunosuppressant having calcineurin-inhibiting activityxe2x80x9d means a whole blood or plasma sample obtained from a patient to whom the immunosuppressant having calcineurin-inhibiting activity has been administered. The sample is preferably subjected to SepPak column treatment as described in EP-0293892-A2, for instance, or pretreated with an extractant such as dichloromethane or methanol.
The complex obtained is separated by an appropriate method and then assayed in the conventional manner taking advantage of any component labeled with a radioisotope or enzyme or using an antibody recognizing some site of any of the components of the complex, whereby the target immunosuppressant can be quantified.
More specifically, the present invention can be practiced in such a manner as mentioned below. Hereinafter, the xe2x80x9cimmunosuppressant having calcineurin-inhibiting activityxe2x80x9d is referred to xe2x80x9cimmunosuppressantxe2x80x9d for short. (1) One of the complex-constituting components, appropriately selected, is fixed or bound to a solid phase such as a plate, test tube, bead or the like in the conventional manner so that the complex obtained can remain fixed to the solid phase. This makes it easy to remove the reaction mixture containing unnecessary components etc. after complex formation.
The complex-constituting component which is to be appropriately selected for binding to the solid phase is the immunophilin, calcineurin or calmodulin.
(1-1): Thus, for example, an excessive amount of an immunophilin fixed to a solid phase, and calmodulin, calcium ion and calcineurin, each in an excessive amount, are reacted with the assay target immunosuppressant contained in the sample, whereby a complex is formed in an amount corresponding to the immunosuppressant content. The complex is separated by suction of the reaction mixture and washing with an appropriate buffer solution, for instance. Then, the complex, hence the complex-forming immunosuppressant, can be assayed using an appropriate enzyme-labeled antibody which recognizes calcineurin or calmodulin.
As the xe2x80x9cenzyme-labeled antibody which recognizes calcineurin or calmodulinxe2x80x9d, there may be mentioned an anti-calcineurin antibody or anti-calmodulin antibody bound, in the conventional manner, to an enzyme generally used in enzyme immunoassay (e.g. peroxidase, xcex2-D-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholine esterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, urease, etc.).
The complex recognized by such an enzyme-labeled antibody as mentioned above can be quantified by assaying the enzyme activity of the label enzyme by the conventional method described in EP-0293892-A2, for instance. Thus, when the label enzyme is peroxidase, for instance, an enzyme substrate solution containing o-phenylenediamine and hydrogen peroxide is used and the color intensity is measured, whereby the complex can be assayed. When the enzyme is alkaline phosphatase, 4-methylumbelliferyl phosphate is used as the enzyme substrate.
(1-2): It is also possible to use, in lieu of the xe2x80x9cenzyme-labeled antibody recognizing calcineurin or calmodulinxe2x80x9d, an antibody recognizing calcineurin or calmodulin (first antibody) and an enzyme-labeled antibody-recognizing said first antibody (second antibody).
As the anti-calcineurin or anti-calmodulin antibody to be used in assay method (1-1) usable as the above first antibody, there is a polyclonal antibody or a monoclonal antibody prepared in the conventional manner using calcineurin or calmodulin as an antigen [e.g. J. Neurochem., 58 (5), 1643-1651 (1992)]. It is possible, however, to use a commercially available anti-calcineurin antibody (=anti-protein phosphatase 2B) or anti-bovine calmodulin antibody marketed by Upstate Biotechnology Co. Ltd. While the class of the antibody is not limited to any particular one, the IgG class is-preferred and an antibody obtained by immunization of mice or the like can be used.
Any polyclonal or monoclonal antibody capable of recognizing the first antibody can be used as the second antibody mentioned above, with such an enzyme as mentioned above under (1-1) bound thereto in the conventional manner. For instance, alkaline phosphatase-labeled anti-rabbit IgG is commercially available and can be purchased, for example, from Vector Laboratories (USA), and it can be used as the labeled second antibody.
(1-3): It is further possible to quantify the complex formed corresponding to the quantity of the immunosuppressant present in the sample, in the conventional manner with enzyme activity or radioactivity as an index, by using, in lieu of ordinary calcineurin or calmodulin, calcineurin or calmodulin labeled by a conventional method with an appropriate enzyme such as mentioned above or an radioisotope (e.g. 125I), in the step of causing complex formation as mentioned under (1-1) following excess immunophilin binding to a solid phase.
(1-4): Calcineurin or calmodulin, each in excess, may be first fixed to a solid phase, in lieu of the immunophilin. In that case, the immunophilin and calcium ion, each in excess, and the sample are added, and complex formation and separation are effected in the same manner as mentioned above. Then, the complex can be assayed using an xe2x80x9cantibody labeled with an appropriate enzyme and capable of recognizing the immunophilinxe2x80x9d (enzyme-labeled anti-immunophilin antibody) prepared by a conventional method. Alternatively, a first antibody recognizing the immunophilin and an enzyme-labeled second antibody capable of recognizing said first antibody may be used as mentioned under (1-2).
(2) On the other hand, it is possible to separate and recover the complex formed, without immobilizing any of the complex-forming components. Thus, the complex formed can be precipitated and separated by reacting therewith an antibody capable of recognizing one of the complex-forming components.
(2-1): For example, the complex can be precipitated and separated using an antibody recognizing calcineurin or calmodulin. In that case, the assay is possible when a certain definite amount of the immunosuppressant labeled with a radioisotope such as 3H is caused to be simultaneously present.
(2-2): On the other hand, the complex can be precipitated and separated using a polyclonal or monoclonal antibody, which recognizes the immunophiline, prepared as described, for example, in International Patent Application WO 94/04700. In that case, calcineurin or calmodulin to be used for complex formation is labeled with an appropriate enzyme or radioisotope (e.g. 125I) as in the case of (1-3) so that the complex can be assayed by a conventional method.
Said-appropriate enzyme to be used for labeling in the above methods (2-2), (1-3) and (1-4) may be any of those described under (1-1).
While various assay methods are conceivable as mentioned above, a preferred quantitive method of immunosuppressant comprises reacting the immunophilin bound in excess to a solid phase with calcineurin, calmodulin and calcium ion, each in excess as well as the immunosuppressant in the sample to thereby cause formation of a complex in an amount corresponding to the amount of the immunosuppressant and, after separation treatment, assaying the calcineurin constituting the thus-obtained complex by an enzyme immunoassay technique. In that case, the enzyme immunoassay technique is preferably one using an anti-calcineurin antibody labeled with an enzyme in standard use, such as peroxidase or alkaline phosphatase, or one using two antibodies, namely an anti-calcineurin antibody and an anti-IgG antibody labeled with such an enzyme as mentioned above.
The assay method of the present invention can be practiced by using either automatic or nonautomatic (manual) means.
Furthermore, a convenient kit can be provided for practicing said assay method. The kit may include all or some of an immunophilin, calcium ion, calmodulin, calcineurin and the immunosuppressant as a standard. It may further include, when necessary, the immunosuppressant labeled with a radioisotope, calcineurin or calmodulin labeled with an appropriate enzyme or radioisotope, an anti-immunophilin, anti-calcineurin or anti-calmodulin antibody labeled with an appropriate enzyme, an anti-immunophilin antibody, an anti-calcineurin antibody, an anti-calmodulin antibody, and/or the like.
On the occasion of complex formation, the calcium ion is used in ordinary cases. However, it is also possible to use any other ion if it can participate in complex formation together with calcineurin, calmodulin and the immunosuppressant. Thus, for instance, the manganese ion, which contributes to similar complex formation as established in Biochemical and Biophysical Research Communications, 192 (3), 1388-1394 (1993), can be used as an alternative.
Furthermore, the complex comprising an immunophilin, calcineurin, calmodulin, calcium ion and the immunosuppressant having calcineurin-inhibiting activity may further have any other component(s) added or attached thereto provided that said components will not disturb the separation and assay following complex formation.